Method and system of microfluidic immunoassay using magnetic beads

ABSTRACT

A microfluidic Western blot method and system including a microfluidic western blot method for immunoassay of proteins, the method including introducing a sample including the proteins onto a chip; electrophoretically separating the proteins; binding the separated proteins to beads to form protein-attached beads, the beads being magnetic; flowing the protein-attached beads into a magnetic holding region; applying a magnetic field to the magnetic holding region to fix the protein-attached beads in place within the magnetic holding region; binding primary antibodies to target proteins on the protein-attached beads; binding secondary antibodies to the bound primary antibodies; and detecting the bound secondary antibodies.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 61/927,960, filed Jan. 15, 2014, incorporated herein inits entirety by reference.

TECHNICAL FIELD

The technical field of this disclosure is assay systems, particularly,methods and systems of microfluidic immunoassay using magnetic beads.

BACKGROUND OF THE INVENTION

The use of microfluidic technology has been proposed for a number ofanalytical chemical and biochemical operations. This technology allowsone to perform chemical and biochemical reactions, macromolecularseparations, and the like, that range from the simple to the relativelycomplex, in easily automated, high-throughput, low-volume systems.Further information about microfluidic devices and systems is presentedin U.S. Pat. No. 6,534,013 to Kennedy, issued Mar. 18, 2003, andincorporated in its entirety herein by reference.

As used herein, the term “microfluidic,” or the term “microscale” whenused to describe a fluidic element, such as a passage, chamber orconduit, generally refers to one or more fluid passages, chambers orconduits which have at least one internal cross-sectional dimension,e.g., depth or width, of between about 0.1 μm and 500 μm. In the devicesof the present invention, the microscale channels preferably have atleast one cross-sectional dimension between about 0.1 μm and 200 μm,more preferably between about 0.1 μm and 100 μm, and often between about0.1 μm and 20 μm.

In general, microfluidic systems include a microfluidic device, or chip,that has networks of integrated submicron channels in which materialsare transported, mixed, separated, and detected. Microfluidic systemstypically also contain components that provide fluid driving forces tothe chip and that detect signals emanating from the chip.

Microfluidic chips may be fabricated from a number of differentmaterials, including glass or polymeric materials. An example of acommercially available microfluidic chip is the DNA LabChip®manufactured by Caliper Life Sciences, Inc. of Hopkinton, Mass., andused with the Agilent 2100 Bioanalyzer system manufactured by AgilentTechnologies, Inc. of Palo Alto, Calif. The chip has two majorcomponents: a working part made of glass, and a plastic caddy or mountbonded to the working part. The working part contains microfluidicchannels in its interior, and wells on its exterior that provide accessto the microfluidic channels. The working part is typically fabricatedby bonding together two or more planar substrate layers. Themicrofluidic channels in the working part are formed when one planarsubstrate encloses grooves formed on another planar substrate. The mountprotects the working part of the chip, and provides for easier handlingof the chip by a user. The increased ease of handling partially resultsfrom the fact that the mount is larger than the working part of thedevice, which in many cases is too small and thin to be easily handled.The mount may be fabricated from any suitable polymeric material, suchas an acrylic or thermoplastic. The glass working part is typicallybonded to the polymeric mount using a UV-cured adhesive. Reservoirs inthe mount provide access to the wells on the working part of the chip.The reservoirs hold much greater volumes of material than the wells inthe working part, thus providing an interface between themacro-environment of the user and the microenvironment of the wells andchannels of the microfluidic device.

This type of microfluidic chip is a “planar” chip. In a planar chip, theonly access to the microchannels in the chip is through the reservoirsin the caddy and in-turn through the wells in the working part. Anothertype of microfluidic chip is a “sipper” chip, which has a small tube orcapillary (the “sipper”) extending from the chip through which fluidsstored outside the chip can be directed into the microfluidic channelsin the chip. Typical sipper chips have between one and twelve sippers.In use, the sipper is placed in a receptacle having sample material andminute quantities of the sample material are introduced, or “sipped”through the capillary tube to the microfluidic channels of the chip.This sipping process can be repeated to introduce any number ofdifferent sample materials into the chip. Sippers make it easier tocarry out high-throughput analysis of numerous samples on a singlemicrofluidic chip.

Western blot electrophoresis assays have been developed to detectspecific proteins in a sample. The process can be divided into threeparts: protein separation, sample transfer, and immunoassay. In proteinseparation, mechanical and/or chemical techniques are applied to asample, such as a tissue sample, to expose proteins. The proteins arethen separated with gel electrophoresis in which the speed of movementof the different proteins through the gel under a differential voltageis governed by the molecular weight of the individual proteins. Insample transfer, the separated proteins are moved from within the gelonto a membrane in a process called electroblotting, which uses electriccurrent to move the proteins. In immunoassay, a primary antibody isattached to target proteins on the membrane, a secondary antibody isattached to the primary antibody, and a light emitter reacts with thesecondary antibody to produce light at each of the target proteins.Detection of the light provides identification and quantification of thetarget proteins.

Although the current method of Western blot electrophoresis assayprovides valuable results, the current method has a number of problems.The current method is a labor-intensive process, performed manually andrequiring gel plates and special membrane paper to transfer theseparated proteins. The manual nature of the process increases the costand limits the number of samples which can be tested. A typical Westernanalysis requires between 8 and 24 hours of monitored operation, withalmost half requiring hands-on, manual operation.

It would be desirable to have methods and systems of microfluidicimmunoassay using magnetic beads that would overcome the abovedisadvantages.

SUMMARY OF THE INVENTION

One aspect of the invention provides a microfluidic Western blot methodfor immunoassay of proteins, the method including introducing a sampleincluding the proteins onto a chip; electrophoretically separating theproteins; binding the separated proteins to beads to formprotein-attached beads, the beads being magnetic; flowing theprotein-attached beads into a magnetic holding region; applying amagnetic field to the magnetic holding region to fix theprotein-attached beads in place within the magnetic holding region;binding primary antibodies to target proteins on the protein-attachedbeads; binding secondary antibodies to the bound primary antibodies; anddetecting the bound secondary antibodies.

Another aspect of the invention provides a microfluidic western blotmethod for immunoassay of proteins, the method including providing amicrofluidic chip having a substrate defining a sample well, aseparation region operably coupled to the sample well, and a magneticholding region operably coupled to the separation region; introducing asample including the proteins into the sample well; flowing the sampleinto the separation region; applying a voltage across the separationregion to electrophoretically separate the proteins in the separationregion; binding the electrophoretically separated proteins to beads toform protein-attached beads, the beads being magnetic; flowing theprotein-attached beads into the magnetic holding region; applying amagnetic field to the magnetic holding region to fix theprotein-attached beads in place within the magnetic holding region;binding primary antibodies to target proteins on the protein-attachedbeads; binding secondary antibodies to the bound primary antibodies; anddetecting the bound secondary antibodies.

Another aspect of the invention provides a microfluidic Western blotsystem for immunoassay of proteins with beads, the system including amicrofluidic chip having a substrate defining a sample well, aseparation region operably coupled to the sample well, and a magneticholding region operably coupled to the separation region; and anelectromagnet operably connected to provide a magnetic field to themagnetic holding region, the magnetic field being operable to fix thebeads in place within the magnetic holding region.

Another aspect of the invention provides a microfluidic method forimmunoassay of analytes, the method including resolving in a first fluidregion one or more analytes in a sample disposed within the first fluidregion based on size and charge of the one or more analytes; binding theresolved analytes to magnetic beads to form analyte-attached beads;applying a magnetic field to fix at least a portion of theanalyte-attached beads in place; binding a detection reagent to theanalyte-attached beads; and detecting the detection reagent.

The foregoing and other features and advantages of the invention willbecome further apparent from the following detailed description of thepresently preferred embodiments, read in conjunction with theaccompanying drawings. The detailed description and drawings are merelyillustrative of the invention, rather than limiting the scope of theinvention being defined by the appended claims and equivalents thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic top view of one embodiment of a microfluidicWestern blot device with multiple sample wells, separate destain andbeads wells, and a detection reagent well made in accordance with theinvention.

FIG. 2 is a schematic top view of one embodiment of a microfluidicWestern blot device with a combined destain and bead well made inaccordance with the invention.

FIG. 3 is a schematic top view of one embodiment of a microfluidicWestern blot device with multiple sample wells and a combined destainand bead well made in accordance with the invention.

FIG. 4 is a schematic top view of one embodiment of a microfluidicWestern blot device with multiple sample wells and separate destain andbead wells made in accordance with the invention.

FIG. 5 is a schematic top view of one embodiment of a microfluidicWestern blot device with multiple sample wells, a combined destain andbead well, and a detection reagent well made in accordance with theinvention.

FIG. 6 is a schematic top view of one embodiment of a microfluidicWestern blot device with a sample sipper made in accordance with theinvention.

FIG. 7 is a schematic top view of one embodiment of a microfluidicWestern blot device with an antibody sipper made in accordance with theinvention.

FIG. 8 is a flow chart of a microfluidic Western blot method inaccordance with the invention.

Like elements share like reference numbers between and among the variousfigures.

DETAILED DESCRIPTION

FIG. 1 is a schematic top view of one embodiment of a microfluidicWestern blot device with multiple sample wells, separate destain andbeads wells, and a detection reagent well made in accordance with theinvention.

The microfluidic chip 100 has a substrate 102 which defines a number ofwells and channels for performing a Western blot method of immunoassay.In this embodiment, the substrate 102 defines a sample well 110, aseparation region 120 operably coupled to the sample well 110, and amagnetic holding region 130 operably coupled to the separation region120.

The sample well 110 can be connected to the separation region 120 by asample channel 111. The sample well 110 can also be operably connectedto an injection electrode 116 through an injection channel 117 throughthe sample channel 111. In operation, the sample including the proteinscan be flowed from the sample well 110 through the sample channel 111into the injection channel 117 by applying a differential voltagebetween the sample well 110 and the injection electrode 116. Theinjection channel 117 can be operably connected to the first separationelectrode channel 119. The sample can be flowed from the injectionchannel 117 to the first separation electrode channel 119 by applying adifferential voltage between the injection electrode 116 and firstseparation electrode 118 operably connected to the first separationelectrode channel 119. In one embodiment, the differential voltage usedto move the proteins around the chip can be a high differential voltage,in contrast with a low differential voltage which can be used to moveimmunoassay chemicals through the magnetic holding region.

In this example, the microfluidic chip 100 includes a number of samplewells 110, 112, 114. The samples from the sample wells can be processedsequentially, i.e., the sample from sample well 110 can be processedfirst, followed by the sample from the sample well 112 and the samplewell 114. Those skilled in the art will appreciate that the microfluidicchip 100 can include any number of sample wells as desired for aparticular application.

The first separation electrode channel 119 can be operably connected tothe separation region 120. The separation region 120 is primed with geland dye so that electrophoresis can be performed on the sample in theseparation region 120. The separation region 120 can also be connectedto a second separation electrode 140 through the magnetic holding region130 and second separation electrode channel 141.

Electrophoresis can be performed on the sample by applying adifferential voltage between the first separation electrode 118 and thesecond separation electrode 140. Electrophoresis separates the proteinsin the sample into one or more protein peaks as the lower molecularweight proteins move more quickly than the heavier molecular weightproteins through the gel of the separation region 120 between thedownstream end 121 and the upstream end 123. The differential voltagebetween the first separation electrode 118 and the second separationelectrode 140 can also be used to move the sample from the separationregion 120 to the magnetic holding region 130.

A destain well 150 can be operably connected to the downstream end 123of the separation region 120 through a destain channel 151 to adddestaining solution to the separated sample leaving the separationregion 120. The destaining solution removes detergent (SDS) micelles toallow visualization of protein peaks in the sample and reduce signalbackground. The destaining solution can be flowed into the sample byapplying a differential voltage between an electrode associated with thedestain well 150 and the second separation electrode 140.

A peak detection region 170 can be provided between the separationregion 120 and the magnetic holding region 130. A peak optical detector(not shown) monitoring the peak detection region 170 can detect theprotein peaks in the sample moving through the peak detection region170, which can be used to detect when the last protein peak enters themagnetic holding region 130.

A bead well 160 can be operably connected to the downstream end of thepeak detection region 170 through a bead channel 161 to add beads to theseparated sample leaving the peak detection region 170. The surfaces ofthe beads are functionalized to attach to any and all proteins in thesample to form protein-attached beads. Further, the beads are magneticand can be magnetically manipulated within the magnetic holding region130. In one embodiment, the beads can be primary antibody attachedbeads, i.e., a bead with a primary antibody attached to the bead at thetime of manufacture and before the bead is introduced onto the chip.Exemplary beads are available from PerkinElmer chemagen Technologie GmbHof Baesweiler, Germany. In one embodiment, the beads can be nanobeads.The beads can be flowed into the sample by applying a differentialvoltage between an electrode associated with the bead well 160 and thesecond separation electrode 140.

An immunoassay channel 181 can be attached downstream of the beadchannel 161 and before the magnetic holding region 130 to allow additionof immunoassay chemicals. In this example, the immunoassay channel 181is operably connected to a blocking buffer well 180, a primary antibodywell 190, a secondary antibody well 200, and a detection reagent well210. When differential voltage between the first separation electrode118 and the second separation electrode 140 is used to move the sampleinto the magnetic holding region 130, the differential voltage can beturned off before the immunoassay chemicals are added.

In operation, when the last protein peak enters the magnetic holdingregion 130, the differential voltage between the first separationelectrode 118 and the second separation electrode 140 can be turned offand an electromagnet (not shown) operably connected to the magneticholding region 130 can be energized to fix the protein-attached beads inplace within the magnetic holding region 130. In one example, themagnetic field is generated by a circular electromagnet maintaining theprotein-attached beads dispersed across the capillary section within themagnetic holding region 130.

The immunoassay chemicals from each of the blocking buffer well 180,primary antibody well 190, secondary antibody well 200, and detectionreagent well 210 can be flowed through the magnetic holding region 130to contact the protein-attached beads in turn by applying a differentialvoltage between an electrode associated with each of the wells and thesecond separation electrode 140. The magnetic holding region 130 can bewashed between application of each of the immunoassay chemicals asdesired for a particular application by applying a differential voltagebetween an electrode associated with the destain well 150 and the secondseparation electrode 140. Each of the immunoassay chemicals can beallowed to incubate within the magnetic holding region 130 to provide adesired incubation time and/or temperature as desired for a particularapplication by removing the differential voltage between the electrodeassociated with each of the wells and the second separation electrode140 after one of the immunoassay chemicals has been flowed into themagnetic holding region 130.

A blocking buffer can be flowed into the magnetic holding region 130 byapplying a differential voltage between an electrode associated with theblocking buffer well 180 and the second separation electrode 140. Theblocking buffer is used after protein binding to the beads to saturateall remaining protein binding sites of the beads and preventnon-specific immunoassay reagents binding to the beads. Those skilled inthe art will appreciate that the immunoassay can be performed withoutuse of a blocking buffer as desired for a particular application. Anyunbound blocking buffer can be washed from the magnetic holding region130 by applying a differential voltage between the electrode associatedwith the destain well 150 and the second separation electrode 140.

A primary antibody can be flowed into the magnetic holding region 130 byapplying a differential voltage between an electrode associated with theprimary antibody well 190 and the second separation electrode 140. Theprimary antibody binds with target proteins on the protein-attachedbeads. Any unbound primary antibody can be washed from the magneticholding region 130 by applying a differential voltage between theelectrode associated with the destain well 150 and the second separationelectrode 140. In one example, the microfluidic chip 100 includes aheating element (not shown) operably connected to the magnetic holdingregion 130 to incubate the primary antibody on the protein-attachedbeads at a temperature as desired for a particular application.

A secondary antibody can be flowed into the magnetic holding region 130by applying a differential voltage between an electrode associated withthe secondary antibody well 200 and the second separation electrode 140.The secondary antibody binds with the primary antibody bound to theprotein-attached beads. Any unbound secondary antibody can be washedfrom the magnetic holding region 130 by applying a differential voltagebetween the electrode associated with the destain well 150 and thesecond separation electrode 140.

A detection reagent can be flowed into the magnetic holding region 130by applying a differential voltage between an electrode associated withthe detection reagent well to 10 and the second separation electrode140. The detection reagent reacts with the secondary antibody bound tothe primary antibody, which is bound to the target protein. In oneexample, the secondary antibody includes a coupled enzyme (such ashorseradish peroxidase HRP, for example) and the detection reagent is anenzyme substrate (such as horseradish peroxidase tyramide signalamplification HRP/TSA, for example) which reacts with the coupled enzymeand generates light.

An immunoassay optical detector (not shown) can be used to detect lightfrom the secondary antibodies. In one embodiment, the immunoassayoptical detector is operably connected to receive light from themagnetic holding region 130 when the protein-attached beads are fixed inplace within the magnetic holding region 130. The magnetic field in themagnetic holding region 130 can be released after the light is measuredand the sample can be removed from the microfluidic chip 100. In anotherembodiment, the immunoassay optical detector is operably connected toreceive light from the protein-attached beads as the protein-attachedbeads flow past the immunoassay optical detector within the magneticfield in the magnetic holding region 130 has been released and adifferential voltage has been applied between the first separation theelectrode 118 and the second separation electrode 140.

A waste well can be associated with the second separation electrode 140so that the sample can be removed from the microfluidic chip 100 byapplication of a differential voltage between the first separation theelectrode 118 and the second separation electrode 140. In oneembodiment, another sample, such as a sample from the second sample well112, can be tested after the first sample is removed from the chip. Inanother embodiment, another sample, such as a sample from the secondsample well 112, can be moved into the separation region 120 at the sametime that the first sample is being removed from the chip.

Those skilled in the art will appreciate that the microfluidic chip 100can be adapted as desired for a particular application. In oneembodiment, one or more of the separation region 120, the peak detectionregion 170, and/or the magnetic holding region 130 is a channel. Inanother embodiment, one or more of the separation region 120, the peakdetection region 170, and/or the magnetic holding region 130 is achamber. The driving force moving the sample through the microfluidicchip 100 can be differential voltage and/or differential pressure alongthe channels. The microfluidic chip 100 can be adapted for use inperforming other types of immunoassays.

The immunoassay chemicals can also be selected as desired for aparticular application. In one embodiment, the primary antibody bindswith a single target protein and the immunoassay optical detectorreceives light at a single wavelength to identify and quantify thesingle target protein. In another embodiment, multiplexing can beperformed on a single chip, where the primary antibody is a mixture ofantibodies that bind with different target proteins and are associatedwith different secondary antibodies. The difference secondary antibodiescan generate light at different wavelengths, so that more than onetarget protein can be identified and quantified at one time whenreceiving light from the magnetic holding region at the immunoassayoptical detector.

FIGS. 2-7 illustrate various combinations of the elements of differentmicrofluidic Western blot devices. Those skilled in the art willappreciate that the various elements can be provided in differentcombinations as desired for a particular application.

FIG. 2 is a schematic top view of one embodiment of a microfluidicWestern blot device with a combined destain and bead well made inaccordance with the invention. In this embodiment, the microfluidic chip300 has a substrate 302 which forms a destain/bead well 350 operablyconnected to the downstream end 123 of the separation region 120 througha destain/bead channel 351 to add a mixture of the destaining solutionand beads to the separated sample leaving the separation region 120. Themixture of destaining solution and beads can be used to wash theimmunoassay chemicals (blocking buffer, primary antibodies, secondaryantibodies) from the magnetic holding region. This embodiment includes asingle sample well 110 rather than multiple sample well and omits thedetection reagent well connected to the immunoassay channel 181.

FIG. 3 is a schematic top view of one embodiment of a microfluidicWestern blot device with multiple sample wells and a combined destainand bead well made in accordance with the invention. In this embodiment,the microfluidic chip 400 has a substrate 402 which forms a destain/beadwell 350 operably connected to the downstream end 123 of the separationregion 120 through a destain/bead channel 351 to add a mixture of thedestaining solution and beads to the separated sample leaving theseparation region 120. This embodiment includes a multiple sample wells110, 112, 114 and omits the detection reagent well connected to theimmunoassay channel 181.

FIG. 4 is a schematic top view of one embodiment of a microfluidicWestern blot device with multiple sample wells and separate destain andbead wells made in accordance with the invention. In this embodiment,the microfluidic chip 500 has a substrate 502 which forms multiplesample wells 110, 112, 114. This embodiment includes a separate destainwell 150 and bead well 160, to avoid bead in the peak detection region170 and beads in the destaining solution used to wash the immunoassaychemicals (blocking buffer, primary antibodies, secondary antibodies)from the magnetic holding region. This embodiment also omits thedetection reagent well connected to the immunoassay channel 181.

FIG. 5 is a schematic top view of one embodiment of a microfluidicWestern blot device with multiple sample wells, a combined destain andbead well, and a detection reagent well made in accordance with theinvention. In this embodiment, the microfluidic chip 600 has a substrate602 which forms multiple sample wells 110, 112, 114. This embodimentincludes a destain/bead well 350 operably connected to the downstreamend 123 of the separation region 120 through a destain/bead channel 351to add a mixture of the destaining solution and beads to the separatedsample leaving the separation region 120. This embodiment also includesthe detection reagent well to 10 operably connected to the immunoassaychannel 181.

FIG. 6 is a schematic top view of one embodiment of a microfluidicWestern blot device with a sample sipper made in accordance with theinvention. In this embodiment, the microfluidic chip 700 has a substrate702 which forms a low-pressure port 710 operably connected to the samplechannel 111 by a low-pressure port channel 711 and a sipper port 720operably connected to the sample channel 111 by a sipper port channel721. In operation, the low-pressure port 710 is held at a lower pressurethan the sipper port 720, which has been introduced into a sample wellof a well plate (not shown), such as a 96-well plate or the like. Thesample contained in the well plate is drawn into the sample channel 111through the sipper port 720. In this example, multiple samples from thewell plate can be processed through the microfluidic chip 700 using thesame immunoassay chemicals from the blocking buffer well 180, theprimary antibody well 190, and the secondary antibody well 200.

FIG. 7 is a schematic top view of one embodiment of a microfluidicWestern blot device with an antibody sipper made in accordance with theinvention. In this embodiment, the microfluidic chip 800 has a substrate802 which forms a low-pressure port 810 operably connected to theimmunoassay channel 181 by a low-pressure port channel 811 and a sipperport 820 operably connected to the immunoassay channel 181 by a sipperport channel 821. In operation, the low-pressure port 810 is held at alower pressure than the sipper port 820, which has been introduced intoan antibody well of a well plate (not shown), such as a 96-well plate orthe like. The antibody contained in the well plate is drawn into theimmunoassay channel 181 through the sipper port 820. In this example,multiple antibodies from the well plate can be processed through themicrofluidic chip 800 using the same sample from the sample well 110.

FIG. 8 is a flow chart of a microfluidic Western blot method inaccordance with the invention. The microfluidic Western blot method 900for immunoassay of proteins includes introducing a sample 910 includingthe proteins onto a chip; electrophoretically separating the proteins920; binding the separated proteins to beads 930 to formprotein-attached beads, the beads being magnetic; flowing theprotein-attached beads into a magnetic holding region 940; applying amagnetic field to the magnetic holding region 950 to fix theprotein-attached beads in place within the magnetic holding region;binding primary antibodies to target proteins 960 on theprotein-attached beads; binding secondary antibodies to the boundprimary antibodies 970; and detecting the bound secondary antibodies980. The method 900 can be performed using a microfluidic chip having asubstrate defining a sample well, an separation region operably coupledto the sample well, and a magnetic holding region operably coupled tothe separation region, as illustrated in FIGS. 1-7.

Referring to FIG. 8, introducing a sample 910 including the proteinsonto a chip moves the sample into position to perform the Western blotmethod. In one embodiment, the sample can be loaded by applying adifferential voltage between electrodes on the chip. In anotherembodiment, the sample can be loaded by applying a differential pressurebetween ports on the chip. Those skilled in the art will appreciate thatthe sample can be loaded in any way desired for a particularapplication. In one embodiment, the method 900 can include priming thechip with gel and dye before the introducing 910.

Electrophoretically separating the proteins 920 can include applying adifferential voltage to separate the proteins in the sample into one ormore protein peaks as the lower molecular weight proteins move morequickly than the heavier molecular weight proteins through the gel of aseparation region on the chip.

Binding the separated proteins to beads 930 to form protein-attachedbeads attaches substantially all of the separated proteins in the sampleto the beads. The beads are magnetic, so the protein-attached beads canbe moved by a magnetic field. In one embodiment, the binding theseparated proteins to beads 930 can include destaining the proteinsbefore binding the separated proteins to the beads. Theelectrophoretically separating the proteins 920 can then further includedetecting migrating peaks in the destained proteins. The flowing theprotein-attached beads into a magnetic holding region 940 can thenfurther include flowing the protein-attached beads into the magneticholding region until a last one of the migrating peaks is detected atwhich point all of the protein-attached beads will be in the magneticholding region.

Flowing the protein-attached beads into a magnetic holding region 940places the protein-attached beads in position in the magnetic holdingregion for immunoassay. In one embodiment, the protein-attached beadscan flow from applying a differential voltage between electrodes on thechip. In another embodiment, the protein-attached beads can flow fromapplying a differential pressure between ports on the chip. Thoseskilled in the art will appreciate that the protein-attached beads canbe made to flow into a magnetic holding region in any way desired for aparticular application.

Applying a magnetic field to the magnetic holding region 950 to fix theprotein-attached beads in place within the magnetic holding region holdsthe protein-attached beads in place during the immunoassay. In oneexample, the magnetic field from an electromagnet can hold theprotein-attached beads to the base of the magnetic holding region. Themagnetic field also can preserve the relative position of the proteinpeaks of the sample within the magnetic holding region.

Binding primary antibodies to target proteins 960 on theprotein-attached beads tags the target proteins for detection whileleaving proteins which are not of interest untagged. In one embodiment,the binding primary antibodies to target proteins 960 can includeincubating the primary antibodies on the protein-attached beads andwashing unbound primary antibodies from the magnetic holding region. Inanother embodiment, the method 900 can include flowing blocking bufferthrough the magnetic holding region over the protein-attached beadsbefore the binding primary antibodies. The method 900 can then furtherinclude incubating the blocking buffer on the protein-attached beads andwashing unbound blocking buffer from the magnetic holding region.

Binding secondary antibodies to the bound primary antibodies 970 tagsthe bound primary antibodies with the secondary antibodies to be used inidentifying the bound primary antibodies, which are attached to thetarget proteins. In one embodiment, the binding secondary antibodies tothe bound primary antibodies 970 can include washing unbound secondaryantibodies from the magnetic holding region.

Detecting the bound secondary antibodies 980 can provide an indicationof the target proteins in the sample, since the bound secondaryantibodies are attached to the bound primary antibodies, which areattached to the target proteins. In one embodiment, the method 900includes flowing detection reagent through the magnetic holding regionover the protein-attached beads before the detecting 980 and thedetecting the bound secondary antibodies 980 includes detecting lightemitted from reaction of the detection reagent with the bound secondaryantibodies.

The detecting the bound secondary antibodies 980 can be performed withthe sample in the magnetic holding region or as the sample flows fromthe magnetic holding region. In one embodiment, the detecting 980 caninclude detecting the bound secondary antibodies in the magnetic holdingregion with the magnetic field applied to the magnetic holding region.In another embodiment, the method 900 can include releasing the magneticfield in the magnetic holding region to release the protein-attachedbeads. The detecting 980 can then include detecting the bound secondaryantibodies flowing by a stationary detector.

The method 900 can continue with emptying the magnetic holding regionand/or introducing a new sample for analysis. In one embodiment, themethod 900 can include releasing the magnetic field in the magneticholding region to release the protein-attached beads. The method 900 canfurther include introducing a second sample onto the chip and performinga Western blot analysis on the second sample as desired. In oneembodiment, the second sample can be tested after the first sample isremoved from the chip. In another embodiment, the second sample can beelectrophoretically separated at the same time that the first sample isbeing removed from the chip. Washes of the chip can be provided betweensequential samples to prevent cross contamination as desired for aparticular application.

It is important to note that FIGS. 1-8 illustrate specific applicationsand embodiments of the invention, and are not intended to limit thescope of the present disclosure or claims to that which is presentedtherein. Upon reading the specification and reviewing the drawingshereof, it will become immediately obvious to those skilled in the artthat myriad other embodiments of the invention are possible, and thatsuch embodiments are contemplated and fall within the scope of thepresently claimed invention. The examples above deal primarily withWestern blot immunoassay of proteins on a chip using magnetic nanobeads,but those skilled in the art will appreciate that the method and systemof microfluidic immunoassay using magnetic beads can be applied equallywell to immunoassay of any analytes on a chip using magnetic beads.

While the embodiments of the invention disclosed herein are presentlyconsidered to be preferred, various changes and modifications can bemade without departing from the spirit and scope of the invention. Thescope of the invention is indicated in the appended claims, and allchanges that come within the meaning and range of equivalents areintended to be embraced therein.

1. A microfluidic western blot method for immunoassay of proteins, themethod comprising: introducing a sample including the proteins onto achip; electrophoretically separating the proteins; binding the separatedproteins to beads to form protein-attached beads, the beads beingmagnetic; flowing the protein-attached beads into a magnetic holdingregion; applying a magnetic field to the magnetic holding region to fixthe protein-attached beads in place within the magnetic holding region;binding primary antibodies to target proteins on the protein-attachedbeads; binding secondary antibodies to the bound primary antibodies; anddetecting the bound secondary antibodies.
 2. The method of claim 1further comprising priming the chip with gel and dye before theintroducing.
 3. The method of claim 1 wherein the binding the separatedproteins to beads further comprises destaining the proteins beforebinding the separated proteins to the beads.
 4. The method of claim 3wherein the electrophoretically separating further comprises detectingmigrating peaks in the destained proteins.
 5. The method of claim 4wherein the flowing further comprises flowing the protein-attached beadsinto the magnetic holding region until a last one of the migrating peaksis detected.
 6. The method of claim 1 further comprising flowingblocking buffer through the magnetic holding region over theprotein-attached beads before the binding primary antibodies.
 7. Themethod of claim 6 further comprising incubating the blocking buffer onthe protein-attached beads and washing unbound blocking buffer from themagnetic holding region.
 8. The method of claim 1 wherein the bindingprimary antibodies further comprises incubating the primary antibodieson the protein-attached beads and washing unbound primary antibodiesfrom the magnetic holding region.
 9. The method of claim 1 wherein thebinding secondary antibodies further comprises washing unbound secondaryantibodies from the magnetic holding region.
 10. The method of claim 1further comprising flowing detection reagent through the magneticholding region over the protein-attached beads before the detecting, thedetecting further comprising detecting light emitted from reaction ofthe detection reagent with the bound secondary antibodies.
 11. Themethod of claim 1 wherein the detecting comprises detecting the boundsecondary antibodies in the magnetic holding region with the magneticfield applied to the magnetic holding region.
 12. The method of claim 1further comprising releasing the magnetic field in the magnetic holdingregion to release the protein-attached beads.
 13. The method of claim 12further comprising flowing the protein-attached beads from the magneticholding region.
 14. The method of claim 12 wherein the detectingcomprises detecting the bound secondary antibodies flowing by astationary detector.
 15. The method of claim 12 further comprisingintroducing a second sample onto the chip.
 16. A microfluidic Westernblot method for immunoassay of proteins, the method comprising:providing a microfluidic chip having a substrate defining a sample well,a separation region operably coupled to the sample well, and a magneticholding region operably coupled to the separation region; introducing asample including the proteins into the sample well; flowing the sampleinto the separation region; applying a voltage across the separationregion to electrophoretically separate the proteins in the separationregion; binding the electrophoretically separated proteins to beads toform protein-attached beads, the beads being magnetic; flowing theprotein-attached beads into the magnetic holding region; applying amagnetic field to the magnetic holding region to fix theprotein-attached beads in place within the magnetic holding region;binding primary antibodies to target proteins on the protein-attachedbeads; binding secondary antibodies to the bound primary antibodies; anddetecting the bound secondary antibodies.
 17. A microfluidic Westernblot system for immunoassay of proteins with beads, the systemcomprising: a microfluidic chip having a substrate defining: a samplewell; a separation region operably coupled to the sample well; and amagnetic holding region operably coupled to the separation region; andan electromagnet operably connected to provide a magnetic field to themagnetic holding region, the magnetic field being operable to fix thebeads in place within the magnetic holding region.
 18. The system ofclaim 17 further comprising a heating element operably connected to themagnetic holding region to control temperature for incubation within themagnetic holding region.
 19. The system of claim 17 wherein thesubstrate further defines a peak detection region between the separationregion and the magnetic holding region and the system further comprisesa peak optical detector operable to detect protein peaks in the peakdetection region.
 20. The system of claim 17 further comprisingimmunoassay optical detector operably connected to receive light fromthe magnetic holding region.
 21. A microfluidic method for immunoassayof analytes, the method comprising: resolving in a first fluid regionone or more analytes in a sample disposed within the first fluid regionbased on size and charge of the one or more analytes; binding theresolved analytes to magnetic beads to form analyte-attached beads;applying a magnetic field to fix at least a portion of theanalyte-attached beads in place; binding a detection reagent to theanalyte-attached beads; and detecting the detection reagent.
 22. Themethod of claim 17 wherein the one or more analytes comprise proteins.23. The method of claim 22 wherein the resolving compriseselectrophoretically separating the proteins.
 24. The method of claim 21further comprising introducing the sample including the one or moreanalytes into the first fluid region.
 25. The method of claim 21 whereinthe applying the magnetic field further comprises flowing theanalyte-attached beads into a second fluid region.
 26. The method ofclaim 25 wherein the second fluid region comprises a magnetic holdingregion.
 27. The method of claim 21 wherein: the binding the detectionreagent further comprises binding primary antibodies to theanalyte-attached beads; and binding secondary bodies to the boundprimary antibodies; and the detecting the detection reagent furthercomprises detecting the bound secondary antibodies.
 28. The method ofclaim 27 further comprising flowing the analyte-attached beads to asecond fluid region.
 29. The method of claim 21 wherein: the beadscomprise primary antibody attached beads; and the binding the detectionreagent further comprises binding secondary antibodies to the primaryantibody attached beads.